The invention relates to the field of cell type-specific gene expression.
Dopamine xcex2-hydroxylase (DBH) is a hallmark protein of noradrenergic neurons because noradrenaline is synthesized by this enzyme. The highly restricted pattern of DBH expression in the nervous system predicts that this gene is subject to neuron-specific as well as to cell type-specific control mechanisms. Transgenic mice experiments have shown that 5.8 or 4 kb of the 5xe2x80x2 flanking sequences of the human DBH gene can drive expression of the reporter gene in neurons of the locus coeruleus as well as other noradrenergic neurons and adrenal chromaffin cells, albeit with some ectopic expression. More recently, comparison of reporter gene expression in transgenic animals generated by using DBH 5xe2x80x2 flanking regions of different lengths indicated that the upstream region between xe2x88x921.1 and xe2x88x920.6 kb is necessary for expression in adult and fetal noradrenergic Neurons. Using cell culture systems, we and others have demonstrated that the 5xe2x80x2 upstream region of the DBH gene can drive reporter gene expression in a cell-specific manner.
Deletional and site-directed mutational analyses have indicated that as little as 486 bp of the upstream sequence of the human DBH gene can direct expression of a reporter gene in a cell type-specific manner. In the 486 bp region of the human DBH gene, the distal part spanning xe2x88x92486 to xe2x88x92263 bp appears to have a cell-specific silencer function which contributed to suppression of the promoter activity in nonneuronal cells. Transient transfection assays identified the proximal part spanning xe2x88x92262 to +1 bp as sufficient and essential for the high-level DBH promoter activity in DBH-positive cells. In this 262 bp proximal area, four protein-binding regions (domains I to IV) have been identified by DNase I footprinting analysis. A cAMP response element (CRE), 5xe2x80x2-TGACGTCC-3xe2x80x2 (SEQ ID NO: 3), with a single base deviation from the consensus octamer motif, 5xe2x80x2-TGACGTCA-3xe2x80x2 (SEQ ID NO: 4), was shown to be critical for both the basal and cAMP-inducible transcription in DBH-expressing cell lines. This CRE is included in a composite enhancer domain structure located at xe2x88x92185 to xe2x88x92150 bp, designated domain IV, which contains several additional cis-elements such as AP1, YY 1, and two core motifs of homeodomain (HD) binding sites. Site-directed mutagenesis of each sequence motif has revealed that the CRE is essential for basal promoter activity in every cell line, YY1 is multifunctional, and the AP1-like motif may be transcriptionally inactive.
The murine paired-like HD protein, Phox2a, is selectively expressed in noradrenergic cells and is critical for development of several noradrenergic neuron populations, including the locus coeruleus. The forced expression of Phox2a robustly activates DBH promoter activity, strongly suggesting a mechanism for noradrenergic-specific promoter function. Moreover, Phox2b, which contains an HD identical to that of Phox2a, has been identified and shown to be widely coexpressed with Phox2a in both the central and peripheral nervous system. Cotransfection assays showed that Phox2a and Phox2b transactivate the DBH promoter activity with a comparable efficiency.
We have discovered that an expression construct that included multiple copies of noradrenergic-specific enhancer domains isolated from the DBH gene increased the minimal promoter activity by 100- to 200-fold in DBH-positive cell lines. Moreover, we discovered that this expression construct maintained the cell-type specificity exhibited by the natural DBH promoter.
Accordingly, the invention features an enhancer cassette having the formula [X-Y]n, wherein each X is independently a noradrenergic cell-specific enhancer derived from a DBH gene; Y is absent or is a mono or polynucleotide that has between one and thirty nucleotides; and n is an integer between three and fifty, inclusive. Preferably, the noradrenergic cell-specific enhancer binds specifically to Phox 2a, Phox2b, or both. Also preferably, X is independently selected from the group consisting of 5xe2x80x2-GTGTCATTAGTGCCAATTAGAG-3xe2x80x2 (SEQ ID NO: 1) and 5xe2x80x2-CCGCTAGACAAATGTGATTACC-3xe2x80x2 (SEQ ID NO: 2); Y is absent or is a mono or polynucleotide that has between one and six nucleotides; and n is between three and twenty, inclusive. In other embodiments, X includes a region that shares greater than 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2 and binds to Phox2a or Phox2b.
The enhancer cassette is useful for expressing a nucleic acid molecule in a noradrenergic cell. To this end, the enhancer cassette can be combined with an RNA polymerase binding site and a transcription initiation site to form an expression construct. Additionally, the enhancer cassette and expression construct of the invention can each be a component of an expression vector, such as an adenoviral vector.
As used herein, by xe2x80x9cnucleic acidxe2x80x9d is meant either DNA or RNA. A xe2x80x9cnucleic acid moleculexe2x80x9d may be a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Unless otherwise specified, the left hand direction of the sequence of a single-stranded nucleic acid molecule is the 5xe2x80x2 end, and the left hand direction of double-stranded nucleic molecule is referred to as the 5xe2x80x2 direction.
By xe2x80x9cpromoterxe2x80x9d is meant a region of nucleic acid, upstream from a translational start codon, which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cDBH promoterxe2x80x9d is one derived from the promoter region of a DBH gene and that, when operably linked to a heterologous nucleic acid molecule, is capable of initiating transcription of that molecule when present in a transcription medium capable of supporting transcription.
Exemplary transcription media include, for example, a mammalian cell (e.g., an immortalized cell), and a yeast cell. Also included are in vitro expression systems such as reconstituted expression medium composed of components required to support transcription, as are known in the art.
By xe2x80x9cenhancer domainxe2x80x9d or xe2x80x9cdomainxe2x80x9d is meant a nucleic acid sequence that, when positioned proximate to a promoter and present in a transcription medium capable of supporting transcription, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain. By xe2x80x9cenhancer cassettexe2x80x9d is meant a nucleic acid sequence that includes an enhancer domain and, optionally, additional sequence that does not enhance transcription (e.g., spacer sequence).
By xe2x80x9cmultimerized enhancer domainxe2x80x9d is meant two or more copies of a noradrenergic cell-specific enhancer domain derived from a DBH gene. Preferably, the number of copies is between three and twenty, inclusive. The enhancer domains can be in the same or opposite orientation, and can be contiguous or noncontiguous. In expression constructs having two different enhancer domains (e.g., domain A and domain B), the orientation and the 5xe2x80x2 to 3xe2x80x2 order (e.g., 5xe2x80x2-AABB-3xe2x80x2 vs. 5xe2x80x2-ABAB-3xe2x80x2) are not limitations to the invention.
By xe2x80x9coperably linkedxe2x80x9d is meant that a nucleic acid molecule to be transcribed and an expression construct (i.e., a promoter and an enhancer domain) are connected in such a way as to permit transcription of the nucleic acid molecule in a suitable transcription medium.
By xe2x80x9cderived fromxe2x80x9d is meant that a the nucleic acid molecule was either made or designed from a second nucleic acid molecule, the derivative retaining important functional features of the nucleic acid molecule from which it was made or designed. In the case of enhancer domains, the important features are specific binding to Phox2a and/or Phox2b and conferral of noradrenergic cell-specific expression when operably linked to a promoter. Optimization of binding and/or cell-specific expression may be performed.
By xe2x80x9cexpression constructxe2x80x9d is meant a nucleic acid molecule that is capable of directing transcription. An expression construct of the present invention includes, at the least, a multimerized DBH enhancer domain and a promoter. Additional domains, such as a transcription termination signal, may also be included, as described herein.
By xe2x80x9cvectorxe2x80x9d or xe2x80x9cexpression vectorxe2x80x9d is meant an expression system (e.g., an adenoviral expression system), a nucleic acid-based shuttle vehicle, a nucleic acid molecule adapted for nucleic acid delivery, or an autonomous self-replicating circular DNA (e.g., a plasmid). When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell""s nucleus or cytoplasm.
By xe2x80x9cplasmidxe2x80x9d is meant an autonomous DNA molecule capable of replication in a cell, and includes both expression and nonexpression types.
By xe2x80x9cheterologousxe2x80x9d is meant that the nucleic acid molecule originates from a foreign source or, if from the same source, is modified from its original form. Thus, a xe2x80x9cheterologous promoterxe2x80x9d is a promoter not normally associated with the multimerized enhancer domain of the present invention. Similarly, a heterologous nucleic acid molecule that is modified from its original form or is from a source different from the source from which the promoter to which it is operably linked was derived.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of a nucleic acid molecule (for example, DNA) which is inserted by artifice into a cell, and becomes part of the organism (integrated into the genome or maintained extrachromosomally) which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof.